Molecular Biology / Molecular Diagnostics
Nucleic acid basics
DNA is double-stranded with A-T and G-C pairing; RNA is single-stranded with uracil. PCR requires template, primers, dNTPs, thermostable polymerase, and buffer; cycling involves denaturation, annealing, and extension. Each cycle doubles target, so 30 cycles give roughly a billion-fold amplification.
Platforms
Real-time PCR measures fluorescence each cycle; the cycle threshold (Ct) is inversely proportional to starting target, enabling quantitation such as viral load. Melt curves distinguish products. Reverse transcription PCR detects RNA viruses. Sanger and next-generation sequencing identify mutations (e.g., EGFR variants); FISH and karyotype detect structural changes; microarrays detect copy-number changes.
Controls and pitfalls
Every run needs positive and negative controls plus an internal control to detect inhibition. Specimen inhibitors include heme, heparin, and high protein. Amplicon contamination causes false positives; prevent it with separate rooms, UNG, and unidirectional workflow. EDTA whole blood is preferred for germline testing; sodium heparin inhibits PCR.
Traps
- Reading a higher Ct as a higher viral load (it is lower).
- Using a qualitative assay to monitor viral load.
- Ignoring a mutation reported at low allele frequency.
Example: Ct 20 versus Ct 30 - the Ct 20 sample contains roughly 1000-fold more target, assuming full amplification efficiency.